The phosphorylation of protein has been proposed to be one of the molecular mechanisms involved in cellular regulation. In this investigation, two different experimental preparations have been used to study the effects of ethanol on protein phosphorylation. These include the cholinergic synaptosomes and synaptosomal plasma membranes from squid optic lobe and synaptosomes and synaptosomal plasma membrane (SPM) derived from rat brain. The protein phosphorylation in cholinergic synaptosomes was stimulated by acetylcholine and calcium. The addition of low concentrations of ethanol (10-50 mM) caused an increase in synaptosomal protein phosphorylation and a decrease in SPM protein phosphorylation. Similar results were obtained in synaptosomes and SPM derived from rat brain. The effect of ethanol on phosphorylating activity of cAMP dependent protein kinase (catalytic subunit) has been studied using specific substrates. Ethanol (10-500 mM) caused a concentration dependent decrease in phosphorylating activity of catalytic subunit. Patterns of protein phosphorylation have been characterized in cytosolic proteins isolated from ethanol treated rat brains. Certain brain regions (e.g. hippocampus, caudate nuclei) showed a decrease in the amount of one phosphorylated high molecular weight (greater than 200,000 dalton) protein during ethanol dependence and withdrawal. This information provides a biochemical basis for the investigation of altered central nervous system biochemistry which could be associated with changes in neuronal excitability characteristics of alcohol dependence and withdrawal.